The enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes and regulates the survival and metastasis of cancer cells. EZH2 is the catalytic subunit of the Polycomb Repressor Complex 2 (PRC2) which functions to silence target genes by tri-methylating lysine 27 of histone H3 (H3K27me3). Histone H3 is one of the five core histone proteins involved in the structure of chromatin in eukaryotic cells. While the precise mechanism by which aberrant EZH2 activity leads to cancer progression is not fully understood, many EZH2 target genes are tumor suppressors suggesting that loss of tumor suppressor function plays an important role.
EZH2 is overexpressed in aggressive solid tumors, including the prostate, breast, skin, bladder, liver, pancreas, and head and neck cancers. For instance, EZH2 transcript and protein were shown to be consistently elevated in invasive breast carcinoma compared with normal breast epithelia. Tissue microarray analysis, which included 917 samples from 280 patients, demonstrated that EZH2 protein levels were strongly associated with breast cancer aggressiveness. Overexpression of EZH2 in immortalized human mammary epithelial cell lines promotes anchorage-independent growth and cell invasion. EZH2-mediated cell invasion required an intact SET domain and histone deacetylase activity (Kleer et al, (2003) Proc. Natl Acad. Sci USA 100(20):11606-11611).
The expression and function of EZH2 in cancer cell lines are inhibited by microRNA-101 (miR-101). An analysis of human prostate tumors revealed that miR-101 expression decreases during cancer progression, paralleling an increase in EZH2 expression. One or both of the two can genomic loci encoding miR-101 were somatically lost in 37.5% of clinically localized prostate cancer cells (6 of 16) and 66.7% of metastatic disease cells (22 of 33). There also is a greater risk of recurrence after prostatectomy in tumors expressing high levels of EZH2 (Varambally et al, (2008) Science 322:1695-1699).
Somatic activating and inactivating mutations of EZH2 have been reported. Somatic activating mutations in EZH2 have been identified in follicular lymphoma and diffuse large B-cell lymphomas that result in increased levels of H3K27me3 (for a review, see Helin and Dhanak (2013) Nature 502:480-488; Pasqualucci et al, (2011) Nature Genet. 43(9):830-837). These mutations taken together with overexpression of EZH2 in various solid tumors suggests that misregulation of EZH2 can lead to silencing of genes that are important to tumor growth and survival. Interestingly, however, inactivating mutations of EZH2 have been identified in myleodysplastic syndrome (MDS) suggesting a potential tumor suppressor role of EZH2 as well.
Thus, enhanced EZH2 activity contributes to undesired cellular proliferation and invasiveness of tumor cells, in part, through trimethylation of H3K27. Since increased H3K27me3 levels appear to contribute to cancer aggressiveness in many tumor types, the inhibition of EZH2 activity may provide therapeutic benefit for a wide range of cancers. The compounds of the present invention offer potential therapeutic benefit as inhibitors of EZH2 activity that may be useful for negatively modulating the activity of EZH2 in a cell or for treating various forms of cancer.